Transplantation of allogeneic cells, tissues and organs is an evolving therapy that has become an increasingly attractive therapeutic option. The number of patients receiving transplants from unrelated donors is expected to double in the near future. Alloreactivity after transplantation has a major impact on clinical outcome, with pathological as well as beneficial effects. HLA mismatches are known to induce an immune reaction after transplantation, however the factors involved in predicting risk of unwanted immune reaction are not well understood.
Hematopoietic Stem Cell Transplantation
Hematopoietic Stem Cell Transplantation (HSCT) is one example of a quickly growing therapeutic option. The major limiting factor of HSCT remains graft-versus-host disease (GVHD), and since the number of patients receiving HSCT is expected to increase, the provision of novel approaches to prevent GVHD must be accelerated. To overcome the risk of GVHD, patients are preferably transplanted with a donor that is completely matched for all HLA-alleles. However, due to diversity of HLA molecules in the population, these completely matched donors are not available for approximately 40% of patients. When a completely matched donor is not available, a clinician often has to face the difficult decision to choose the best donor out of the mismatched donors (i.e. the one that carries the lowest GVHD risk). Until now, determining which donor is most suitable relies on a laborious assay that requires up to 14 days of labwork.
Historically, alloreactivity after HSCT is considered to be evoked mostly due to direct recognition of HLA disparities by donor T cells. This means that the graft T cells recognize mismatched HLA that is expressed as an intact molecule on the cell surface of host cells. The present invention is based in particular upon indirect recognition. Alloreactivity can be evoked when peptides derived from the mismatched host HLA allele are processed and presented on shared HLA and thereby recognized by the donor T cells.
Alloreactivity after hematopoietic stem-cell transplantation (HSCT) has a major impact on clinical outcome, with pathological as well as beneficial effects. The pathological effect of alloreactivity is reflected by graft-versus-host disease (GVHD). The risk of acute GVHD (aGVHD) is dependent on the level of matching for the HLA-A, -B, -C, -DRB1, and -DQB1 alleles, with an optimal match being a full match for five loci (a 10/10 match).
Recipients of matched-unrelated HSCT have a 24% reduced cumulative incidence of severe aGVHD compared to recipients of single mismatched donors (Ref 1a). Although recipients of 10/10-matched HSCT have a 47% increased hazard ratio (HR) for leukaemia relapse when compared to HSCT with donors mismatched for one HLA-C allele (Ref 2a), transplant protocols preferentially select 10/10-matched donors as overall survival is significantly inferior in partially matched-unrelated donors (Ref 1a). However, fully matched-unrelated donors are not available for all patients; in 40% of the situations, a single HLA-mismatched donor (a 9/10 match) is the best available alternative (Ref 3a).
In these situations, definition of the best-permissible mismatch may help prevent GVHD and, subsequently, inferior outcome. Recently, certain specific non-permissible mismatches have been identified on an epidemiological basis, in relation to an increased risk of developing severe aGVHD (Ref 4a). The mechanism underlying the increased risk of GVHD after HSCT with these non-permissible mismatches remains poorly characterised.
Functionally, better-permissible mismatches are determined with cytotoxic T-lymphocyte precursor frequency (CTLpf) assays. CTLpf above 1/105 are predictive for developing severe aGVHD (Ref 5a). Moreover, CTLpf less than or equal to 1 per 106 PBL is associated with a better overall survival. (Ref: Heemskerk et al (2007) Bone Marrow Transplantation, 40, 193-200) Thus, these criteria can be used to distinguish between the potentially mismatched donors. However, the CTLpf assay is laborious, delays time to transplantation, and is therefore not used in most transplant centres. Additionally, materials from potential donors need to be shipped and tested before use in transplantation. There are presently no effective means available for direct donor selection pre-delivery, based on eliminating samples which have a likelihood producing a negative result.
To find an alternative for the CTLpf assay, multiple, so far unsuccessful, attempts have been undertaken to predict non-permissible mismatches using two generally available prediction programs, HLAMatchmaker and HistoCheck. HLAMatchmaker determines potential epitopes for antibodies and has proven its validity for solid-organ transplantation (Ref 6a, 7a). HLAMatchmaker considers differences in amino-acid triplets as epitopes on HLA. Although antibodies potentially play a role in the development of GVHD, predictions based on HLAMatchmaker are not correlated to alloreactivity (Ref 1a). HistoCheck is based on the concept of direct recognition of HLA disparities, that is, donor T cells recognize an intact mismatched-HLA molecule loaded with a non-polymorphic peptide (Ref 9a). HistoCheck determines the structural differences in HLA molecules in the peptide-binding grooves or regions contacting the T-cell receptor (Ref 10a). By determining these structural differences, it aims to predict the likelihood of direct recognition of HLA disparities. Dissimilarity scores obtained with HistoCheck are also not correlated to alloreactivity (Ref 11a, 12a).
In light of the previously existing techniques there exists a need for more reliable methods for predicting whether donor material for a transplantation, which is HLA mismatched, is at increased risk of leading to a failed transplantation, for example development of GVHD, and/or an increase in mortality.
Kidney Transplantation
Matching for human leukocyte antigens (HLA) significantly improves the outcome of kidney transplantation (reviewed in Ref 1b). However, as a result of the high level of polymorphism of the various HLA loci and the limited number of donors, HLA mismatches between donor and recipient exist in approximately 85% of cadaveric kidney transplantations (Eurotransplant database; http://www.eurotransplant.org, accessed Apr. 24, 2012). Evidently, these HLA mismatches frequently lead to production of HLA-specific antibodies, which shorten graft survival (Ref 2b) and reduce the re-transplantation options. In order to prevent antibody formation against HLA, the optimal kidney grafts are either HLA identical to the recipient, or express acceptable HLA mismatches which do not induce antibody formation. To a limited extent, these acceptable mismatches can be identified with the HLAMatchmaker algorithm.
HLAMatchmaker defines polymorphic epitopes on HLA molecules, called eplets, accessible by HLA antibodies and subtracts those eplets present on the patients' own HLA (Ref 3b, 4b). In case this leaves no eplets to be recognized on a kidney graft, no antibody responses are to be expected (Ref 5b). Although HLAMatchmaker predicts which HLA-antigens can potentially induce HLA antibody formation, it does not predict T-cell reactivity towards allogeneic HLA (Ref 6b).
In previous studies it has been shown that the HLA-DR phenotype of the responder influences the production of Bw4-specific antibodies and class-I antibody sensitization grade (Ref 7b, 8b). This suggests a role for indirect recognition of donor-derived HLA peptides on HLA class-II molecules of the antigen-presenting cells of the patient. This phenomenon would explain Thelper-2-cell responses leading to the production of donor-specific antibodies (DSA) of the IgG isotype (Ref 9b).
Binding of Peptides to HLA Molecules
Binding of peptides to HLA molecules is predictable. The differences between predicted binding affinities and experimental measurements have been shown to be as small as the differences in measurements between different laboratories (Ref 10b). Predictability is particularly high for HLA class-I molecules, as these molecules have a more strict preference for nine amino acid long peptides (9-mers) and require specific amino acids as anchor residues at clearly defined anchor positions (Ref 11b). For HLA class II molecules predictability is lower, as peptides of different length can bind using different positions as anchor residues (Ref 12b). Therefore, it is difficult to determine how a peptide aligns to the HLA class II-binding groove and which amino-acid residues in the peptide are preferred as anchors. To solve this problem, Nielsen et al. used a so-called core predictor to estimate how a peptide positions in the class II binding groove (Ref 23a). The core predictor enabled the development of an accurate HLA class-II predictor, called NetMHCII (Ref 24a).
Despite the advances in predicting peptide binding to class I and class II HLA molecules, there is still significant uncertainty in assessing the factors involved in alloreactivity and the production of donor-specific antibodies (DSA). Considering the tools presently available to predict unwanted immune responses after kidney transplantation, there exists a need to provide more reliable methods for assessing potentially adverse reactions in advance of transplantation.
Indirect Recognition of Mismatched-HLA Antigens
Conceptually, mismatched HLA-directed T-cell alloreactivity may result from direct and indirect recognition of HLA disparities. So far, studies that aimed at explaining and predicting the clinical alloreactivity towards mismatched HLA, mainly focused on direct recognition of HLA disparities. Direct recognition involves donor T cells that recognize an intact mismatched-HLA molecule loaded with a non-polymorphic peptide (Ref 9a). When polymorphisms in HLA alleles lead to differences in the peptide-binding groove, the presented peptide repertoire of HLA molecules may differ substantially. These different peptide repertoires may lead to T-cell responses. The HistoCheck algorithm determines structural differences in HLA molecules in the peptide-binding grooves or regions contacting the T-cell receptor, thereby predicting a dissimilarity score (Ref 10a). However, the scores obtained with HistoCheck do not correlate with alloreactivity, neither in vitro (Ref 6c), nor in vivo (Ref 7c, 11a, 12a).
T-cell related alloreactivity can potentially also be evoked by indirect recognition of the mismatched-HLA allele. Indirect recognition has been studied in great detail for minor histocompatibility (H) antigens. Mismatches for these HLA-presented polymorphic proteins are associated with an increased risk of aGVHD (Ref 13a), and a decreased risk of relapse (Ref 14a, 15a). Analogous to peptides derived from minor H mismatches, peptides derived from mismatched-HLA molecules can also be presented by HLA.
Indirect recognition of the mismatched-HLA antigen may lead to T cell-related alloreactivity. During indirect HLA recognition, T cells recognize peptides derived from polymorphic HLA antigens presented by a shared (matched) HLA molecule. Peptides derived from mismatched-HLA molecules are frequently presented by HLA (Ref 10c). These indirectly recognizable HLA epitopes have been associated with both acute and chronic graft failure in solid organ transplantation (Ref 11c, 12c, 13c, 14c). T cells that indirectly recognize HLA-mismatches in the context of self-HLA may therefore play an important role in clinical alloreactivity.
With approximately 8800 HLA alleles identified today (Ref 15c), experimentally determining all potential HLA-derived epitopes presented in all HLA antigens is an extensive and hardly possible task. To facilitate the identification of these indirectly recognizable HLA epitopes, a novel approach has been developed. This approach is based on validated prediction tools (Ref 18a, 19a, 21a, 20a, 20c).
The present invention designates the HLA-derived epitopes that are predicted to be presented as Predicted Indirectly ReCognizable HLA Epitopes (PIRCHES). The present invention identifies PIRCHES presented by shared—HLA class-I (PIRCHE-I) and class-II (PIRCHE-II) separately. PIRCHE-II are shown to induce alloreactivity after kidney transplantation; PIRCHES presented by HLA-DR correlated with the de novo development of donor-specific HLA IgG antibodies (Ref 21c).
The present invention is therefore based on the finding that recognition of HLA-derived peptides has an effect on clinical alloreactivity after HLA-mismatched HSCT. To this end, numbers of predicted PIRCHE-I and -II can be assessed and their role evaluated in the adverse clinical effects of HSCT. On the basis of such investigation, the present invention describes universally applicable methods that can predict non-permissible HLA mismatches prior to HSCT and other cell or organ transplants.